207
Egypt. J. Agric. Res., 91 (1), 2013
PHYSIOLOGICAL AND HISTOCHEMICAL STUDIES ON THE
LAND SNAIL, MONACHA CARTUSIANA
IN SHARKIA GOVERNORATE
ISMAIL, SH.A.1, A.A.RASHED2, F.M.ABOU-SENNA2 and M. ABED1
1. Plant Protection Research Institute, Agric. Res. Center Dokki, Giza, Egypt
2. Department of Zoology, Faculty of Science, Al Azhar University, Cairo, Egypt
(Manuscript received 3 June 2012)
Abstract
Physiological and histochemical studies were conducted on
Monacha cartusiana snail, in order to determine some physiological
and histochemical aspects during active and aestivation periods.
Results of these studies showed reduction of the percentage of
glucose in aestivation periods compared with the period of activity,
where the concentration of glucose in the activity revealed 52.63
mg / dL, compared with an average concentration in aestivation,
reaching 16.16 mg / dL. Regarding the concentration of protein, it
observed a decrease in aestivation period compared with activity
period, where it reached 3.39 g / dL in the activity period
compared with 1.9 g / dL in aestivation. In case of urea the
opposite results were recorded, where the urea concentration in
the period of activity was 5.97 mg / dL compared with 7.89 mg /
dL in aestivation. The results of histochemical studies indicated the
presence of large amounts of carbohydrates in the cells of the
digestive gland as well as in the cells of foot in the previous of
aestivation. Moreover the study showed that the snail consumed
large amounts of carbohydrates during aestivation period.
INTRODUCTION
Terrestrial molluscs has become one of the serious pests in most of world
countries especially those with moist or rainy climates, they are vegetarian and feed
on a wide variety of plant parts both wild and cultivated that including field vegetable
and fruit crops as well as ornamental plants (Godan, 1983). In Egypt, until 1980, land
snails were considered as dangerous crop pests and cause effective damage in the
northern governorates. In Sharkia Governorate the land snail, Monacha cartusiana
reported a relatively high occurrence and population density on economic crops
(Ismail, 1997). Snails survive many months without food and water under aestivation.
The aestivated snails draw on their reserve of fat and glycogen at much reduced rate
(Akinnusi, 1998). Histochemical investigations (Stickle, 1971 and Veldhuijzen and Van
Beek, 1976) postulated that many molluscs which store glycogen in large amounts
were noticed to utilize it during starvation period as a primary source of energy.
According to (Beddiny 1979) carbohydrates were the first materials in the digestive
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PHYSIOLOGICAL AND HISTOCHEMICAL STUDIES ON THE LAND SNAIL,
MONACHA CARTUSIANA IN SHARKIA GOVERNORATE
gland in the two fresh water snails Helisoma duryi and Physa acuta to be used during
starvation. Haemolymph composed of water, inorganic salts (mostly Na, Cl, K, Mg and
Ca) and organic compounds (mostly carbohydrates, proteins and lipids) (Burton,
1965). There are limits to the duration of aestivation that can be tolerated by land
snails and mortality eventually increases as aestivation is prolonged (Rees and Hand,
1993). Therefore, this study investigated the effect of aestivation period on glucose,
total protein and urea levels in haemolymph and the localization of carbohydrate in
digestive gland and foot of the land snail, Monacha cartusiana during active period
and various period of aestivation. The aim of this study to determine some
physiological and histochemical aspects during activity and aestivation periods.
MATERIALS AND METHODS
I: Physiological studies:This study was conducted to determine the concentration of glucose, protein
and urea during the period of activity in different periods of aestivation (early and
mid, as well as at the end of aestivation) found in Monacha cartusiana snail
haemolymph. For this purpose, 30 adult snails were collected from heavy infested
field at Sharkiat Mubasher village, Al-Ibrahimia country, Sharkia Governorate and then
transferred to laboratory before each period of estimation. The period of estimation
was divided as following: - active season (March, 2010) and in course of dormancy in
the beginning of aestivation (June), mid of aestivation (August) and in the end of
aestivation (October). According to Bezerra et al. (1997) the snails shell was cleaned
with a paper towel and the haemolymph then collected by making a pierce just above
the level of the pericardial cavity, and then a fine micropipette inserted carefully
through a tiny hole and for aspiration of haemolymph. One ml of haemolymph was
obtained from 30 snails in each period. The collected haemolymph was centrifuged at
1000 round per minute (rpm) for 10 minutes in order to precipitate haemocytes and
cellular debris. The resulting supernatant was used in the laboratory analysis for
measurement levels of glucose, total protein and urea according to Jacobs and vanDenmark (1960).
II: Histochemical studies:The present work was carried out to examine the carbohydrate moiety content
in the digestive gland and foot snails such as the stage of normal nutrition compared
to the one that entered in the stage of aestivation. Snails were collected from the
same field mentioned previously at three different times (In active snail (prior to
aestivation), in mid of aestivation and at the end of aestivation). The gastropods mass
ISMAIL, SH.A. et. al.
209
were carefully cleaned with a wet piece of cotton to clean the shell. Then gastropod
mass were individually minced and washed several time in invertebrate saline (0.7%)
to remove mucus debris and pieces of broken shells, then fixed in alcoholic Bouin
solution for 2 hours. To reveal the effect of aestivation on glycogen content of
muscular tissue and hepatopancreas, sections were stained with periodic acid Schiff
(PAS). Sections were then oxidized in 1 % periodic acid for 5 minutes, washed in
running tap water for 3 minutes then stained in Schiff reagent for 15 minutes.
Sections were washed for 10 minutes in running tap water, dehydrated and then
mounted according to Pearse (1972).
RESULTS AND DISCUSSION
I: Physiological studies:The effects of active and aestivation periods on some hemolymph content as
glucose, total protein and urea for M. cartusiana snail were determined under
laboratory conditions. Data presented in Table (1) and Fig (1&2) revealed that glucose
and total protein recorded high levels during active period compared with mean of
levels during aestivation where recorded 52.63 mg/dl and 16.16 mg/dl for glucose,
3.39 and 1.90 g/dl for total protein, respectively. Regarding the values of glucose and
total protein during aestivation, higher values were recorded at the beginning of
aestivation compared with low values at the end of aestivation where recorded 26.03
& 8.46 mg/dl for glucose and 2.23 &1.36 g/dl, respectively.
Interestingly, serum urea exhibited an increase level from the beginning of
aestivation until the end of aestivation showing 3.53 and 10.37 mg/dl, respectively.
Regarding general mean of the three components, it gave 1.9 g/dl for total proteins
and 16.16, 7.89 mg/dl for glucose and urea, respectively. Generally, it could be
concluded that glucose and total proteins compounds were decreased during
aestivation, while urea was increased during aestivation. These results are agree with
those of Friedl, (1975) who indicated that starved snails used protein as an energy
source during starvation. Rees and Hand, (1993) found that in Oreohelix strigosa and
O. subrudis snails polysaccharide was catabolized early in aestivation, and protein was
the primary metabolic substrate after polysaccharide reserves were depleted.
Moreover lipid was catabolized at a low rate throughout aestivation. Regarding urea
and purine bases accumulated during aestivation as a result of protein catabolism.
Abo-Naser, (1998) revealed that higher values in urea concentration found in starved
snails due to the increase in urea cycle activity. Shahawy, (2005) reported that
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PHYSIOLOGICAL AND HISTOCHEMICAL STUDIES ON THE LAND SNAIL,
MONACHA CARTUSIANA IN SHARKIA GOVERNORATE
glucose concentration and total protein in M. cantoina snail decreased in starvation
compared with urea which increased in starvation.
In contrast of these result, Da Silva and Zancan, (1994) found that in
Megalobulimus oblongus snail, the haemolymph glucose levels were higher in summer
than in other seasons. The glycogen content of the hepatopancreas, muscle and
mantle was constant throughout the year, except in winter when lower values were
found. Glycogen metabolism seems to constitute the source of energy substrate
during winter.
Table 1. Biochemical parameters of M. cartusiana snail during active period and
aestivation period.
During active
Glucose (mg/dl)
Total proteins
(g/dl)
Urea
(mg/dl)
Active
Beginning of
Mid of
End of
snails(control)
aestivation
aestivation
aestivation
52.63
26.03
13.99
8.46
16.16
3.39
2.23
2.16
1.36
1.9
5.97
3.53
9.77
10.37
7.89
Glucose (mg/dl)
Total proteins (g/dl)
60
4
3.5
50
Glucose (mg/dl)
Mean
3
40
Total proteins (g/dl)
parameters
During aestivation
period
2.5
30
2
1.5
20
1
10
0.5
0
0
Active
snails(control)
Beginning of
aestivation
Mid of
aestivation
End of estivation
Fig 1. Glucose and Total protein concentration during active period and aestivation
period in M. cartusiana
ISMAIL, SH.A. et. al.
211
Urea (mg/dl)
12
10
8
6
4
2
0
Active
snails(control)
Beginning of
aestivation
Mid of
aestivation
End of
estivation
Fig 2. Urea concentration during active period and aestivation period in M. cartusiana.
II: Histochemical studies:Foot and hepatopancreas of M. cartusiana snail were stained with
heamotoxylin, eosin and periodic acid Shiff (PAS) for the detection on the
carbohydrate content. Experiments results can be summarized in Figs A-F as follows:
A-Hepatopancreas:
Normally, the digestive gland is a large deeply brown colored structure
occupying the visceral hump of the snail. It consists of two unequal lobes where the
anterior part is smaller than the posterior one. The whole gland is enclosed by a
sheath of connective tissue and each lobe is formed of branching tubules which is
surrounded by a thin layer of circular muscle fibers. The cells were arranged around
an irregular lumen and these cells divided into digestive cells, excretory cells and
calcium cells. The digestive cells are formed of columnar or cub shaped the cells
resting on a thin basement membrane. The excretory cells less abundant than
digestive ones they are pyramidal and shorter than digestive cells. Calcium cells low in
number and short present in epithelium base pyramidal in shape.
In active snail (prior to aestivation):
The PAS stained sections showed that, the digestive cells are characterized by
the presence of intensively or deeply stained coarse glycogen granules located mainly
in both the apical and basal region of the cells. The hepatopancreatic acini are filled
with muco poly sacchride (MPS). The secretory bodies exhibit a strong reaction
towards PAS. The irregular lumens are normal in size as illustrated in Plate, (1A).
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PHYSIOLOGICAL AND HISTOCHEMICAL STUDIES ON THE LAND SNAIL,
MONACHA CARTUSIANA IN SHARKIA GOVERNORATE
In mid of aestivation:
Sections stained with PAS showed that the glycogen granules occupied the
apical portion of the digestive cells and showed strongly stained granules of
carbohydrate. The basal regions are moderately colors fine particles Plate, (1B).
At the end of aestivation:
The digestive cells were faintly stained with PAS and glycogen was displayed
as moderately coarse granules in the apical portions of cells and fine particles in the
basal portion. The excretory bodies are occupied with weakly PAS fine granules. The
lumens were reducing in size Plate, (1C).
B- Foot:
The foot is essential powerful muscle, yellow in color, lying beneath the
visceral hump. It consists of an outer epithelial covering represented the mantle and
an inner layer which is dermis consisting of dense fibrous connective tissue. The
epithelium is composed of a single layer of two type of columnar to cuboidal shaped
cells, and ciliated cells distinguished by their apical cilia on surface. It has numerous
secretory cells. The dermis is formed of muscular and collagen fibers interweaved in
all directions among compact reticular connective cells and abundant circular and oval
amebocytes. The muscle cells of the integument are spindle shaped, uninucleate and
unstriated. Muscle cells usually connect directly with the collagenous connective tissue
surrounding them, rather than forming muscle-to-muscle connections. Muscle cells
contain mitochondria in varying numbers, and stored glycogen is present reflecting
the cell’s metabolic demands.
In active snail (prior to aestivation):
The PAS preparations showed that, the foot is characterized by the presence
of deeply stained glycogen granules concentrated on the outer layer (epidermis layer)
and inner layer of foot (dermis and muscle layer) Plate, (1D).
In the mid of aestivation:
The PAS preparation showed mild aggregated of glycogen granules at the
dermis layer of foot. The glycogen granules less than that found in active snail Plate,
(1E).
In end of aestivation:
The PAS preparation showing very little of glycogen granules aggregated at
the edge of foot. The Muscle fibers were free from glycogen granules Plate, (1F).
The
effects
of
aestivation
on
carbohydrate
content
in
foot
and
heapatopancreas of land snail M. cartusiana have been reviewed by many authors.
The obtained results ran parallel with Runham, (1975) found that the digestive gland
is the largest organ in the body of terrestrial gastropods. It consists of two lobes
ISMAIL, SH.A. et. al.
communicating with the stomach via large ducts. Recently, Sharaf, (2009) revealed a
pronounced decline of carbohydrates in the digestive gland cells of snail after
starvation. Severe decline in carbohydrate content was observed especially after 30
days of starvation. Moreover, protein inclusions have exhibited week stain ability in
the digestive gland cells of these snails as a consequence of starvation.
Fig (A): Showing hepatopancreatic acini rich in granules of MPS (actively feeding
snails). L. =lumen, dc.= digestive cell, gl.= glycogen granules, sc.=
secretory cells. X100.
Fig (B): Revealing the hepatopancreas stained with PAS. Note that pyramidal cells are
mildly filled with glycogen granules (gl) with relatively small leumens
(L).X100.
Fig (C): Showing hepaopacreatic acinus (ac) nearly devoides of glycogen granules and
digestive enzymes at the end of aestivation, PAS. X100.
213
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PHYSIOLOGICAL AND HISTOCHEMICAL STUDIES ON THE LAND SNAIL,
MONACHA CARTUSIANA IN SHARKIA GOVERNORATE
Fig (D): Showing glycogen granules (gl) concentrated at the periphery of foot (dark
pinky color), and muscle fibers (mf), PAS.X40.
Fig (E): Showing head foot muscles at the mid time of aestivation showing mild
aggregates of glycogen granules at the periphery of head foot, PAS.X40
Fig (F): Showing very little of glycogen granules aggregated at edges of head foot.
Note the gaps found between bundles of muscle fibers (mf), PAS.X10
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